JPH0460093B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to compositions for normalizing cancer and their use in cancer treatment.

It is a well-known fact that from ancient times until today it has been believed by medical professionals that cancer is an unwanted parasitic neoplasm and therefore must be removed from the human body.

Throughout history, internists have acted along these lines and have therefore performed primary resections.

Along these lines, techniques have been developed to destroy cancer cells starting from the simplest surgical method known as exeresis.

Today, Excellesis continues to be practiced, even if it is not a fully effective technique. In fact, this type of surgery, when used alone, is too often incomplete, and also very often spread from the primary site is inevitable.

Other methods that have been widely practiced are radiotherapy using X-rays, radium, cesium, cobalt, and the like. The principle of this type of treatment is based on the fact that cancer cells are fragile and sensitive to radiation, and although they themselves actively proliferate, they are even more susceptible to being destroyed by radiation.

Unfortunately, however, radiation promotes the recruitment of new cancer cells from normal cells, as was well observed after the explosion of the atomic bomb in Hiroshima.

Finally, much hope is based on the production of new chemical and antimitotic drugs. Antimitotic agents specifically inhibit DNA replication.
It functions at the level of DNA and its expression. Unfortunately, these drugs act interchangeably on both cancer cells and normal cells that undergo rapid proliferation and the regeneration necessary to maintain a healthy body in perfect balance.

Moreover, killing of cancer by chemotherapy results in a very dramatic logarithmic reduction process of cancer cells in the first stage, but does not lead to the killing of all cancer cells.

Finally, antimitotic agents act at the DNA level to specifically inhibit DNA division.
Recently, a size disorder has been revealed that causes mutations, and those with these mutations can develop new cancers years later.

Currently, immunostimulatory therapy is being brought back to the fore.
It is currently being actively researched for the reasons mentioned above.

However, there are other basic problem-solving processes that could not have been considered at a time when the cancer growth process was not understood (and still is not fully understood today). This is so-called "tumor reversion," which involves returning cancer cells to their normal state without destroying them.

This solution is so attractive that when the Griffeul Prize was awarded to UNESCO on October 13, 1981, the normalization of cancer was described as a biological revolution.

In this perspective, recent studies have shown that cancer cells, which were originally normal and therefore non-pathological, have changed in terms of activity for some as yet unknown reason. It is based on the accepted concept that the cells of the body undergo changes that violate the body, leading to a state of disrupted equilibrium and uncontrolled proliferation leading to fatal consequences.

Therefore, by restoring the normal expression of pre-established programs in a healthy body equilibrium, cells that were originally normal but have since transformed into cancerous cells are restored to normal activity.

Researchers are already turning their attention to the normalization of cancer, and are encouraged by the fact that some cancers, although very rare, regress spontaneously.

Many authors have tried to use chemicals that might have a normalizing effect, but without success.

However, after several years of research and experimentation with a wide range of possible normalizing agents, the inventors have determined that rather than using a single type of normalizing agent, they combine the complementary properties of multiple components. We successfully normalized HeLa cells in vitro by using a normalizing composition.

Cancer normalization was demonstrated by two standard criteria well known in the field of cell culture: reproduction of contact inhibition and adhesion to glass.

Uncontrolled growth arrest and reproduction of contact inhibition are
Growth stops when the cell layer in culture spreads over the entire surface of the glass container and the bottom of the glass container acts as a carrier for the cell layer, and as long as this phenomenon is normalized, this phenomenon will be revolutionary. It is true.

Furthermore, normalization of cancer cells is also indicated by good adhesion between cells or between cells and glass.

Cancer cells already have separative properties, which is a major reason why they spread through metastasis.

Since the expression of these two properties was achieved and confirmed by the non-toxic normalizing composition, it was logically imperative to investigate the possibility of using this composition in the medical field.

This composition is very easy to use and effectively inhibits cancer growth. That is, with this composition, the body eliminates by its own natural means (dynamic equilibrium, especially immunological means) cells that were previously dangerous, but are now simply useless.

According to the invention, the composition is characterized in that three chemicals are combined, namely polyethylene glycol, dimethyl sulfoxide and theophylline, simply by mixing in distilled water.

When added to a cancer cell culture (eg, a colony of cloned HeLa cells) in the surrounding medium, this mixture initiates normalization of the cancer.

The following examples describe this composition and its method of use in further detail. However, the scope of the invention is not limited thereto.

Experimental Methods A confluent layer of HeLa cells (cancer cells) in culture was treated according to the present invention.

Culture was carried out in medium (MEM2011 Eurobio) with 10% fetal bovine serum (SVF Eurobio).
It was carried out in a Felcon box with a surface area of 75 cm ² containing 30 ml.

Experiment 1 Basic Experiment Normalization Phenomenon Flask 1 A mixture prepared in the following proportions was added. 1.5 ml of a 10% solution of polyethylene glycol, 0.5 ml of pure dimethyl sulfoxide, and 500 μ of theophylline.

Renewal of the medium was carried out twice every week at the above ratio. Finally, the medium was refreshed without any further addition of normalizing composition.

At the same time as this basic experiment, another flask of the same type was placed for comparison.

Flask 2: Comparison flask without any addition to observe normal growth of HeLa cells in normal medium.

Flask 3 Add only polyethylene glycol (1.5 ml of 10% solution).

Flask 4 Add only pure dimethyl sulfoxide (0.5 ml).

flask 5 A flask containing only 50μ of theophylline.

Flask 6 Add polyethylene glycol without theophylline (1.5 ml of a 10% solution) and pure dimethyl sulfoxide (0.5 ml).

Results Already after 10 hours and 8 days in the flask, regularly arranged cells were observed attached to the glass, while in other parts the original cells continued to be present in a highly irregular state. However, by periodically renewing the medium, the non-normalized cells can be sufficiently removed, displaced by the normal cells, separated, and indeed weakened, to ensure that the non-normalized cells become more rigid over the entire surface of the flask. The normalization process continued from 1 month to 6 weeks later when the tissue spread.

Flask 2 After 3 weeks, the HeLa cell culture became detached and the cells became suspended, as is commonly observed in this type of culture.

Flask 3 The addition of polyethylene glycol did not cause any change in the culture, and the growth progressed the same as in Flask 2.

Flask 4 Addition of dimethyl sulfoxide caused no change in the culture and followed the same growth course as flasks 2 and 3.

Flask 5 The addition of theophylline did not cause any change in the culture and it followed the same growth course as flasks 2, 3 and 4.

Flask 6 However, in this flask, after 6 days, a regularly arranged band of cells that appeared to be undergoing normalization was observed. These cells remained attached to the glass in the middle of the tissue layer in an apparently irregular state.

However, the normalization process did not continue even with further medium renewal. After 6 weeks, the appearance of the culture remained the same (quiescent state) and no matte layer was formed over the entire flask.

Nevertheless, one important property must be noted. Namely, the fact is that although the cultured cells in the comparison flask were left undisturbed for the same time as above, all the cells had already reached a state of suspension during the three week period.

The experiment was terminated after 6 weeks but continued in flask 1. After 18 months, the normalized cells remained in the same state, the so-called quiescent state, remaining in a monolayer and confined at the edge of the flask.

Implementation 2 Comparison of HeLa cells and normalized cells During this basic experiment, HeLa cell layers other than comparison HeLa cells were simply preserved to maintain the culture as a continuous clone.

An unexpected contamination by normalized cells on one of these cell layers actually occurred. Then the actual cell inoculation (like bacterial inoculation) occurs,
After 10 to 15 days, small scattered colonies appeared.

These colonies took up positions at the bottom of the flask and poisoned the culture. These colonies increase in size and form circles from a strictly monolayer of cells, touch each other, and then fuse, spreading over the HeLa cell layer and forming a lip-like bulge upon contact. did.

The HeLa cell layer eventually became mesh-like (wire-mesh-like). The HeLa cells eventually became destroyed, disorganized, disorganized, and in turn suspended. Only a monolayer of normalized cells confined to the rim of the flask remained.

This experiment shows that normalized cells exhibit strong adhesion to glass and superior vigor to HeLa cells in culture (as evidenced by the greater difficulty of trypsinizing these cells). We can conclude that.

As a result of this experiment, which was not originally planned, it was observed that a tissue layer or mat of normalized cells colonizing the entire bottom of the flask exhibited contact inhibition. And it has remained in this static state for the past six months.

Experiment 3: Maintenance of quiescence One of the flasks containing normalized cells was kept in situ for 4 weeks. After 0 hours and 8 months, some of the normalized cells were found to have died, separated, and become suspended, perforating the tissue layer or mat of cells. Moreover, the medium became acidic.
After washing and refreshing, the pine layer was completely reconstituted and the culture returned to its previous quiescent state. 0
After 18 months, the layers were still in the same condition.

Experiment 4 Genetic nature of reproduction of contact inhibition At time 0, one of the flasks containing quiescent normalized cells was subjected to a first passage (this term refers to the same operation as pre-incubation of bacteria). Then, trypsinization was performed.

The cultivation produced new monolayered pine without any difficulties and became quiescent as soon as they reached the rim of the flask.

The second passage took place 23 days later (total period: 23 days).

The third passage took place 26 days later (total period: 49 days).

The fourth passage took place 25 days later (total period: 74 days).

The fifth and final passage was performed 16 days later (total period: 90 days).

An experiment was conducted at the end of the fifth passage with a total duration of 90 days. It was sufficiently demonstrated that the phenomenon was stably reproduced under these experimental conditions.

The experiment was terminated after it was determined that a continuous cloning colony was established and the fact that the normalized HeLa cells had acquired genetic properties was proven.

The normalized cells were then ready to be used in exactly the same way as new serially cloned colony cells needed for other experiments.

Experiment 5 Independence regarding the number of culture updates (from quiescence) To ensure that this last experiment was demonstrable, flasks of normalized cells that had been 18 months old and in quiescence were used.

After trypsinization, new flasks were colonized with inoculated cells. I repeated the passage three times one after another to confirm. Experimental proof of cancer normalization has been confirmed.

This fact was neither natural nor artificial. Furthermore, reproducibility was an established fact.

The above experiment can be summarized as follows.

The Γ normalizing composition acts on HeLa cells in culture to normalize cancer cells.

ΓThis normalization is characterized by the reproduction of important properties that have disappeared due to canceration, in particular: contact inhibition, stronger adhesion to glass, and the ability of cells to form cell tissues to interact with each other. This can be confirmed by the adhesion between the two.

ΓIn particular, contact inhibition is maintained when new mitosis occurs, and therefore this property is genetically transmitted to the next generation, and this depends on the number of culture renewals and the length of the quiescent period. It was clearly shown that it was not controlled by

Γ Furthermore, as clearly shown, at least 2
Two components are essential: polyethylene glycol and dimethyl sulfoxide, and the addition of theophylline makes the normalization process fully effective.

Γ Therefore, the normalization reaction of the composition is a combination of a membrane modifying agent consisting of polyethylene glycol (there is no difference if the molecular weight is between 1000 and 6000) and a normalizing agent consisting of dimethyl sulfoxide to which theophylline is added as an adjuvant. Depends on coordination.

The ratios shown in Γ Experiment 1 are basic and average.

A supplementary experiment established the following.

1 Non-toxic membrane modifier (polyethylene glycol)
The ratio of
(±7%).

2. The proportion of the essentially non-toxic basic normalizing agent (dimethyl sulfoxide) can be increased substantially. It is well known that the diffusion rate of this substance is very high, and this chemical has no viscosity limitations. Therefore, although this ratio can easily be fixed at 20% (±0%) of the volume of the solution, there is virtually no benefit to systematically searching for higher ratios.

3 On the other hand, the proportion of theophylline must be kept within a very narrow range. For 30ml of medium
A fixed proportion of theophylline of 50μ is the experimental standard, and it is not preferable to deviate too much from this, therefore 5% (±2.5%) of the solution.
should be kept within the range of .

Extension of the application of this invention from in vitro to in vivo After obtaining and confirming the results through in vitro testing, it is natural to consider applying it to the medical field as an anticancer agent.

This application must be based on two types of information. These are: 1) toxicological information, and 2) clinical information.

These two points must be successfully addressed in order to realize the potential of using this invention as a medicine on an industrial scale.

1) Toxicological information a) Polyethylene glycol is a neutral chemical substance that does not have any toxicity, has already been used for subcutaneous or intravenous injection, and has no direct (allergenic) effect in either animals or humans. ) has no effect or secondary effects.

b) Dimethyl sulfoxide C ₂ H ₆ SO has been used in very extensive and very varied clinical studies as a result of toxicity studies on a considerable number of animal species and humans. All of these studies are published in the Annual Proceedings of the Academy of Sciences, New York, March 15, 1967, Vol. 141, No. 1, 1-671.
It was published as a 671-page report on
However, this chemical has not been described as a normalizing agent at all. Furthermore, these reports reveal that dimethyl sulfoxide does not have any toxicity.

c) Theophylline C ₇ H ₈ N ₄ O ₂ (dimethyl-1,
3-xanthine) is a chemical that has been used medicinally for many years. This substance is a diuretic, a heart stimulant, and a bronchodilator, especially used against asthma attacks.

Therefore, as far as these three components are concerned, there is no need to conduct further toxicity tests.

However, DL50 will be described next.

Γ Polyethylene Glycol The DL50 of polyethylene glycol (600) in rats is 7.8 g/Kg. This corresponds to 546 g for a human (body weight approximately 70 kg), and the amount sufficient for normalization is considered to be only 1/546 of this DL50.

Γ Dimethyl Sulfoxide The DL50 of dimethyl sulfoxide is 12 g/Kg for mice and 20.5 g/Kg for rats.
Kg. This substance has low systemic toxicity and exhibits the same expression in all species. For comparison, the doses used in humans for a single injection are as follows:
It is 1/1050 of DL50.

ΓTheophylline Theophylline has a DL50 of 400 for mice.
mg/Kg, 300 mg/Kg for rats. These values are high. If we compare this value with the highest dose used in humans, which is 0.02 g of pure theophylline in one injection of 10 ml of the drug, this amount is only 1/1225 of the average DL50 of 350 mg/Kg.

2) Clinical Experiments Although this experiment is the cornerstone to date, a mixture consisting of the following composition: 90 ml of distilled water, 9 g of polyethylene glycol, 9 ml of pure dimethyl sulfoxide, 3 ml of theophylline, usually a 6% pharmaceutical solution, was After administration as a series of subcutaneous injections of 10 ml per dose, generally at 8-day intervals,
The following important observations were obtained.

a) dog Next we got the results.

ΓAfter 3 injections at exceptional intervals of 4-5 days, the skin neoplasms (small secondary white tumors, with a smooth surface and no hair) atrophied, dried up, and 90% of them It disappeared.

Major tumor changes in Γ animals: After 3 injections and 10 injections at 8-day intervals, the large intraocular tumor on the undereye surface that had become undulating became suppurative and fused, resulting in cold abscess ( released outside like cold abscess).

b) Male In the case of leukemia patients, the following results were obtained.

After a few days of Γ-lethal results and two 10 ml injections separated by 24 hours, the number of white colony elements rapidly decreased from 100,000 to 10,000, and the functional value of the remaining elements was maintained.

This functional value is represented by a decrease in blast cells from 92 to 33 and an increase in lymphocytes from 1 to 65. After several more days, blast cells decreased to 7 and lymphocytes progressively increased to 87.

c) Female Γ Breast Cancer - A cancer with a diameter of 7 mm as observed radiographically and echographically (although scientifically desirable, no biopsy was performed to avoid risk to the patient) after 50% of the normalizing composition. It normalized after multiple injections (10 ml subcutaneous injections every 8 days).
The treatment was continued with one injection every 10 days for 2 months after normalization was complete. After normalization, radiographs and echographs were taken once a month. As a result, the image of the cancer itself changed, and there was a central incomplete echo area that was slightly reinforced by posterior echoes compared to the outside. It is shown that

ΓUterine cancer was observed by cervical biopsy as follows. There was a "very large differentiated extracervical infiltration and purulent epidermoid tumor." Treatment was performed with local cesium radiation. An abdominal wall incision was performed. In this case, a biopsy was performed but no resection was performed. Specifically, analysis of the nodules of the iliac bifurcation revealed a large infiltration by numerous neoplastic epidermoid tumors that were poorly differentiated and immature. In the end, the cancer could not be removed because it was present in the bladder and uterus. Normalizing composition 5
Treatment was by multiple injections (10 ml subcutaneously at 8-day intervals). Treatment was continued at the same intervals as above.

As a result, the uterus scan graph revealed that the uterus had an increased volume, a uniform echostructure, and a uniform circumference. In particular, it was reported that ``there is no pelvic mass of note at this time.''
Finally, vaginal application clearly concluded that no cancer was present.

Conclusion The properties of the normalizing composition of this invention were clarified through in vitro experiments, and it was shown that this composition has the same properties in vivo.

ΓThis composition does not have the effect of destroying cancer cells.

ΓThis composition does not actually destroy other cells.

Γ However, once the cancer cells normalize, they remain in place and their presence continues to be demonstrated by radiographs and echographs.

ΓIn any case, it has already been observed in about 20 cases that cell proliferation always stops.

The uncontrolled proliferation caused by Γcancer cells no longer causes harm but the equilibrium action process (lymphocyte action, necrosis or aseptic suppurative fusion)
The host body itself, i.e. its own means (if such means remain) and in particular its immune defense mechanisms, are used to eliminate cells that have to be removed as foreign cells by be exposed.

Experience has shown that in the use of normalizing agents, strict considerations such as those already described and already taken in the field of therapeutic agents must be taken.

However, drug models prepared for in vitro experiments have to be applied to the in vivo field.

For this purpose, physiological serum or Quinton serum is used instead of distilled water.

It is also a well established fact that dilution of polyethylene glycol and dimethyl sulfoxide is effective. On the other hand, an initial injection of a drug containing theophylline reduced to 1/50 and 1/100 of the amount of theophylline already determined and subsequently administered will induce the body to absorb theophylline (a 6% solution in conventional medicine). (known as).

Claims (1)

[Scope of Claims] 1. A cancer normalizing composition comprising polyethylene glycol as a membrane denaturing agent, dimethyl sulfoxide as a basic normalizing compound, and theophylline as an adjuvant. 2. Claim 1, which is obtained by adding 20±10% of pure dimethyl sulfoxide to a 15±7% solution of polyethylene glycol having a molecular weight in the range of 1000 to 6000 in distilled water, and further adding 5±2.5% of theophylline to this mixture. Compositions as described.